PROJECT SUMMARY Sustained availability of efficacious drugs is essential for worldwide efforts to eradicate malaria. The emergence and spread of drug resistance to current antimalarial therapies remains a pressing concern with reports of artemisinin-based treatment failures escalating the need for novel antimalarial chemotherapies. Thus the discovery of new druggable targets and pathways, including those that are critical for multiple life stages, is a major challenge for the development of next-generation therapeutics. Using an integrated chemogenomic approach, we have identified the cytoplasmic prolyl tRNA synthetase in Plasmodium falciparum (PfcPRS) as the long-sought biochemical target of halofuginone. Furthermore, we uncovered an unprecedented mechanism of drug-tolerance in the parasite by modulation of proline homeostasis. In this proposal, we seek to understand the molecular basis of the parasite?s ability to sense and evolve resistance to halofuginone via the Adaptive Proline Response (APR). We bring an integrated approach combining our expertise in molecular parasitology, metabolomics, genetics, and synthetic chemistry to probe these aspects of aminoacyl tRNA synthetase biology and inhibition in the parasite. We will investigate a non-genetic mechanism of resistance to the PfcPRS inhibitor, halofuginone. We identify the primary source of increased intracellular proline in response to halofuginone treatment and strategies to circumvent this process. We will determine if increased proline levels in parasites exhibiting the APR are mediated by changes in key metabolic pathways at the genomic or proteomic level, using high-coverage DNA sequencing and quantitative mass spectrometry-based proteomic technologies. We will explore APR-independent mechanisms of aaRS inhibition in the parasite, evaluating PRS inhibitors with differing binding modes.